WO2002083148A1

WO2002083148A1 - Nerve cell survival promoters containing cyclic phosphatidic acid derivative

Info

Publication number: WO2002083148A1
Application number: PCT/JP2002/003658
Authority: WO
Inventors: Kimiko Murofushi; Gabor Tigyi
Original assignee: Gencom Corporation
Priority date: 2001-04-13
Filing date: 2002-04-12
Publication date: 2002-10-24
Also published as: EP1391204A1; JP2002308778A; JP4836345B2; EP1391204A4; US20040220149A1

Abstract

It is intended to provide novel drugs which are useful in treating and/or preventing nerve disorders by enhancing the survival ratio of nerve cells or promoting the extension of nerve cells. Namely, drugs for promoting nerve cell survival which contain as the active ingredient a cyclic phosphatidic acid derivative having a cyclic phosphate structure at the glycerol sn-2- and 3-positions.

Description

Specification

Neuronal cell survival promoter containing cyclic phosphate derivative

The present invention relates to a drug containing a cyclic phosphatidic acid derivative, which is one of phospholipids. More specifically, the present invention relates to a drug for promoting survival of nerve cells, a drug for promoting elongation of nerve cells, and for treating and / or preventing a neuropathy, which comprises a cyclic phosphatidic acid derivative as an active ingredient. Related to the drug. Background art

Daricellophospholipid, which is the main component of a biological membrane, generally has two molecules of hydrophobic fatty acids bonded to the glycerol skeleton, and a hydrophilic group such as choline ethanolanol is bonded via a phosphate group. . The balance between hydrophobic and hydrophilic moieties in phospholipids is important for forming a stable lipid bilayer. In contrast, lysophospholipids have only one molecule of fatty acid bonded to them, and the hydrophobic part is relatively small relative to the hydrophilic group. Shows active action.

However, in recent years, a large number of lysophospholipids exhibiting specific physiological activities at low concentrations have been found, one of which is lysophosphatidic acid (LPA). LPA is one of the phospholipids with the simplest structure. Phosphatidic acid (PA) is different from phosphatidic acid (PA) in that either the sn_l position or the fatty acid at position 2 of glycerol is desacylated. Different (see Figure 1).

LPA is present in very small amounts (less than 0.5% of total cell phospholipids) in vivo. Traditionally, LPA has been considered an intermediate or degradation intermediate in phospholipid biosynthesis. However, in the late 1970s, plasma (Schumacher, KA, et al., Thromb. Haemostas., 42, 631-640 (1979)) and soybean crude lecithin fraction (Tokumura, et al., Lipids, 13, 468- 472 (1978)), a substance having a vasoconstrictor action was identified as LPA. Furthermore, it has been shown that the lipid growth factor in serum is LPA (van Corven, E., et al., Cell 59, 45-54 (1989)), and LPA has attracted attention as a bioactive lipid. .

LPA has cell growth promoting effects (Fischer DJ, et al., Mol Pharmacol, 54, 979-988 (1988)), and promotes invasion of cancer cells (Imamura, F., et al .: Jpn. J. Cancer Res., 82, 493-496 (1991); Imamura, F., et al .: Biochem. Biophys. Res. Commun., 193, 497-503 (1993); and Imamura, F., et al .: Int. J. Cancer, 65, 627-. 632 (1996)), and inhibition of apoptosis (Umnaky, SR, et al .: Cell Death Diff., 4, 608-616 (1997)), and so on, have been shown so far. In particular, LPA is also known to cause neurite regression in neuronal cells.

(Tigyi, G., et al .: J. Biol. Chem., 267, 21360-21367 (1992); Jalink, K., et al .: Cell Growth & Differ., 4, 247-255 (1994); Jalink, Κ · other:.. J. cell Biol, 126 , 801-810 (1994); and Tigyi, G., other: J. Nurochera, 66, 537-548 ( 1996)) 0 in addition, there is in the nervous system cell line It has also been reported to induce exocytosis in PC12 cells

(Shiono, S., et al .: Biochem. Biophys. Res Commun., 193, 663-667 (1993)) ₀ Furthermore, in 1996, Chun et al. Specifically identified the neuroepithelial cell layer (ventri luar zone, vz). The expressed G protein-related receptor gene (ventriluar zone gene-1; vzg-1 / edg-2) has been cloned, and serum lipids are required for morphological changes of cells overexpressing the gene. The findings revealed that the specific ligand was LPA (Hecht, JH, et al .: J. Cell Biol. 135, 1071-1083 (1996)). These findings suggest the importance of LPA signaling in the nervous system, suggesting that LPA may play an important role in neural development and differentiation.

On the other hand, the present inventors have been carrying out various cell biochemical analyzes using Escherichia coli 7 ^ r polycephaluw as an experimental material. It has been revealed that eukaryotic slime molds undergo morphological changes in response to changes in the external environment, and show significant changes in the composition and metabolism of biomembrane lipids as they proliferate and differentiate. A new lipid component, isolated and identified from the monophasic myxoamoeba in 1992, had a hexapropanoic acid containing a cyclopropane ring at the _Sn -1 position of the glycerol skeleton as a result of structural analysis. -Phosphoric acid rings at 2nd and 3rd position It was confirmed to be a substance having an ester bond in the form (Murakami-Mura of ushi, K., et al .: J. Biol. Chem., 267, 21512-21517 (1992)). This substance was named PHYLPA because it was an LPA analog from Physarum (see Figure 2).

PHYLPA is obtained from a lipid fraction that suppresses the activity of DNA polymerase in eukaryotic cells and suppresses the growth of cultured animal cells.It has been confirmed that PHYLPA exhibits these physiological activities. . PHYLPA has a characteristic fatty acid, but as a result of organic synthesis of structural analogs obtained by substituting this fatty acid moiety with another common fatty acid and examining their physiological activities, it was found that PHYLPA has the same physiological action as PHYLPA. (Murakami-Murofushi, K., et al .: Biochem. Biophys. Acta, 1258, 57-60 (1995)). This suggests that the important structure for these physiological actions is the cyclic phosphate structure at glycerol sn_2-position and 3-position. Lipids with this structure are collectively referred to as cyclic phosphatidic acid (cPA) (see Figure 2).

It was confirmed that cPA is not a lipid unique to Escherichia coli but exists widely in the living world. For example, palmitic acid is included in the fatty acid portion of human serum albumin-bound lipids.

CPA with (C16: 0) was isolated and identified, suggesting that a small amount of cPA bound to myristic acid (C14: 0) and stearic acid (C18: 0) was also present. Concentration of cPA in serum are expected to be about 1 0 one ⁷ M, which corresponds to approximately 1 Z 1 0 serum levels of LPA

(Kobayashi. T., et al .; Life Science, 65, 2185-2191 (1999)). Subsequently, it was confirmed that cPA was also present in human serum and ovine lacrimal gland fluid as well as LPA (Liliom, K., et al .: Am. J. Physiol., 274, C1065-1074 ( 1998)).

It has been reported that the action of cPA also shows a reciprocal or similar physiological activity to that of LPA. For example, suppression of cell proliferation (Murakami-Murofushi, K., et al .: Cell Struct. Funct., 18, 363-370 (1993)), suppression of cancer cell invasion (Mukai, M. et al .: Int. J. Cancer, 81, 918-922 (1999)) and formation of intracellular stress fibers (Fischer, DJ, et al .: Mol. Pharmacol., 54, 979-988 (1998)).

By the way, it is known that nerve cells cannot survive when the supply of neurotrophic factor (nerve growth factor; NGF) is cut off. Exists (Hirata Hatanaka: Protein Nucleic Acid Enzyme, 35, 103-117, 1989). It has also been reported that fibroblast growth factor (FGF), an NGF-like cell growth factor, increases the survival rate of hippocampal neurons and promotes neurite outgrowth (Hirata Hatanaka: Biochemistry). , 61, 1351-1365, 1989). However, the effects of cPA on neurons have not been reported. Disclosure of the invention

The present invention elucidates the effect of cPA on nerve cells as one of the new physiological activities, and is useful for the treatment and / or prevention of neuropathy by improving the survival rate of nerve cells or promoting the elongation of nerve cells. Providing new drugs was an issue to be solved. In order to solve the above problems, the present inventors first attempted to elucidate the mechanism of cPA biosynthesis, and then attempted to detect cPA in the cerebral brain. Using primary cultures derived from rat fetal brain, we analyzed the effects of cPA on neuronal survival and neurite formation. As a result of these analyses, the present inventors demonstrated that cPA enhances the survival rate of rat hippocampal primary cultured nervous system cells and promotes neurite outgrowth. Have been found to be useful therapeutic agents, and have completed the present invention.

That is, according to the present invention, general formula (I):

(In the formula, R is a linear or branched alkyl group having 1 to 30 carbon atoms, a linear or branched alkenyl group having 2 to 30 carbon atoms, or a linear or branched alkyl group having 2 to 30 carbon atoms. Alkynyl groups, and these groups may include a cycloalkane ring or an aromatic ring. M is a hydrogen atom or a counter cation.) Provided as an active ingredient is a cyclic phosphatidic acid derivative represented by the formula (I).

According to another aspect of the present invention, there is provided an agent for promoting elongation of nerve cells, comprising a cyclic phosphatidic acid derivative represented by the above general formula (I) as an active ingredient. According to still another aspect of the present invention, there is provided an agent for treating and / or preventing or treating a neuropathy, comprising as an active ingredient the cyclic phosphatidic acid derivative represented by the above general formula (I).

The neuropathy is selected from, for example, dementia, Alzheimer's disease, senile dementia of the Alzheimer type, amyotrophic lateral sclerosis, Parkinson's disease, stroke, cerebral infarction or head trauma. The cyclic phosphatidic acid derivative represented by the general formula (I) used in the present invention is preferably 1-oleoyl cyclic phosphatidic acid.

According to yet another aspect of the present invention,

A method for promoting the survival of nerve cells, comprising administering a therapeutically effective amount of the cyclic phosphatidic acid derivative represented by the above general formula (I) to a mammal containing human; a therapeutically effective amount of the above general formula ( A method for promoting elongation of nerve cells, which comprises administering a cyclic phosphatidic acid derivative represented by the formula (I) to a mammal containing human; and a therapeutically effective amount of the cyclic phosphatidic acid represented by the above general formula (I). A method for treating and / or preventing a neurological disorder, comprising administering a thidic acid derivative to a mammal containing human.

According to yet another aspect of the present invention,

Use of the cyclic phosphatidic acid derivative represented by the general formula (I) in the manufacture of a medicament for promoting survival of nerve cells;

Use of the cyclic phosphatidic acid derivative represented by the general formula (I) in the manufacture of a medicament for promoting elongation of nerve cells;

And use of the cyclic phosphatidic acid derivative represented by the above general formula (I) in the manufacture of a medicament for treating and / or preventing a neuropathy;

Is provided. BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows the structures of phosphatidic acid (PA) and lysophosphatidic acid (LPA).

FIG. 2 is a diagram showing the structure of a lysophospholipid. A is for one LPA, B is for P

HYLPA, C indicates 1-acyl cPA.

FIG. 3 is a diagram showing an outline of a method for extracting a lipid component from the cerebrum of the Pacific Ocean.

FIG. 4 is a diagram showing a method for purifying cPA by thin layer chromatography (TLC). FIG. 5 is a diagram showing the results of TLC analysis of a partially purified product derived from the cerebrum of the cereal. The area surrounded by the pencil indicates the area detected by the primulin reagent. (A) shows the detection of cPA in the extract, and (b) shows the determination of the cPA concentration in the extract.

FIG. 6 is a view showing neuronal appearance 48 hours after the addition of BSA (a), cPA (b) or NGF (c).

FIG. 7 is a graph showing the effect of cPA on nerve cell density. (A) shows the relationship between cell density and viability, and (b) shows the determination of optimal cell density.

FIG. 8 is a graph showing the effect of cPA on neuronal viability.

FIG. 9 is a diagram showing nerve cells as data for determining an optimal cPA concentration for increasing cell viability.

FIG. 10 is a graph showing the effect of cPA on neurite outgrowth. The vertical axis is 2

The length of control neurons after 4 hours (a) or 12 hours (b) was defined as 1, and the ratio was expressed as a ratio to that.

FIG. 11 is a graph showing the cPA concentration and the percentage of cells having neurites.

FIG. 12 is a diagram of a nerve cell showing the relationship between neurite outgrowth and cPA concentration.

FIG. 13 is a graph showing the effect of a PI3K inhibitor on the survival increasing effect of cPA. Wortmannin (30 nM), LY294002 (10 / zM), c PA (1 μ

M) BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, embodiments of the present invention will be described in detail.

The agent of the present invention can be used for improving the survival rate of nerve cells, promoting the elongation of nerve cells, and treating and / or preventing neurological disorders. The cyclic phosphatidic acid represented by the following general formula (I) can be used. Derivatives are included as active ingredients.

(In the formula, R is a linear or branched alkyl group having 1 to 30 carbon atoms, a linear or branched alkenyl group having 2 to 30 carbon atoms, or a linear or branched alkyl group having 2 to 30 carbon atoms. Alkynyl groups, and these groups may include a cycloalkane ring or an aromatic ring. M is a hydrogen atom or a counter cation.)

In the general formula (I), specific examples of the linear or branched alkyl group having 1 to 30 carbon atoms represented by the substituent R include, for example, methyl group, ethyl group, propyl group, butyl group, pentyl group, Examples include a hexyl group, a heptyl group, an octinole group, a noninole group, a desinole group, a pentadecyl group, an octadecyl group and the like.

Specific examples of the linear or branched alkenyl group having 2 to 30 carbon atoms represented by the substituent R include, for example, an aryl group, a butenyl group, an otenyl group, a decenyl group, a dodecadienyl group, and a hexadecatrenyl group. And more specifically, an 8-decenyl group, an 8-undecenyl group, an 8-dodecenyl group, an 8-tridecenyl group, an 8-tetradecenyl group, an 8-pentadecenyl group, and an 8-hexadecenyl group , 8_heptadecenyl group, 8-octadecenyl group, 8-icosenyl group, 8-docosenyl group, heptade force-8,11-genenyl group, heptadeca 8,11,14-triethyl group, nonadecal 4, 7,10,13-tetraenyl group, nonadhesive 4,7,10,13,16-pentaeynole group, henikosa 3,6,9,12,15,18 — Hexenyl group and the like. Specific examples of the linear or branched alkynyl group having 2 to 30 carbon atoms represented by the substituent R include, for example, 8-decinyl group, 8-pindecinyl group, 8-dodecinyl group, 8-tridecynyl group, —Tetradecynyl group, 8-pentadecyl group, 8—hexadecynyl group, 8-heptadecyl group, 8-otatadecyl group, 8-icoshell group, 8-docosinyl group, heptadeca 8,11-diynyl group and the like.

Specific examples of the cycloalkane ring which may be contained in the above-mentioned alkyl group, alkenyl group or alkynyl group include, for example, a cyclopropane ring, a cyclobutane ring, a cyclopentane ring, a cyclohexane ring, a cyclooctane ring and the like. The cycloalkane ring may contain one or more heteroatoms, and examples thereof include an oxylane ring, an oxetane ring, a tetrahydrofuran ring, and an N-methylprolysine ring.

Specific examples of the aromatic ring which may be contained in the above-mentioned alkyl group, alkenyl group or alkynyl group include, for example, a benzene ring, a naphthalene ring, a pyridine ring, a furan ring and a thiophene ring.

Accordingly, when the substituent R is an alkyl group substituted by a cycloalkane ring, specific examples include a cyclopropylmethyl group, a cyclohexylethyl group, and an 8,9-methanopentadecyl group.

When the substituent R is an alkyl group substituted by an aromatic ring, specific examples include a benzyl group, a phenethyl group, and a p-pentylphenyloctyl group. M in the cyclic phosphatidic acid (cPA) derivative represented by the general formula (I) is a hydrogen atom or a counter cation. Examples of the case where M is a counter cation include an alkali metal atom, an alkaline earth metal atom, and a substituted or unsubstituted ammonium group. Examples of the alkali metal atom include lithium, sodium and potassium, and examples of the alkaline earth metal atom include magnesium and potassium. Examples of the substituted ammonium group include a butylammonium group, a triethylammonium group, and a tetramethylammonium group. As a specific example of cPA represented by the general formula (I) used in the present invention, oleoyl cPA is particularly preferred.

The cPA derivative represented by the general formula (I) is described in, for example, JP-A-5-230888, JP-A-7-149772, JP-A-7-2588278 The compound can be chemically synthesized according to the method described in Japanese Patent Application Laid-Open No. Hei.

Alternatively, the cPA derivative represented by the general formula (I) is synthesized by allowing phospholipase D to act on a lyso-type phospholipid according to the method described in Japanese Patent Application No. 11-37032. You can also. The lyso-type phospholipid used here is not particularly limited as long as it is a lyso-type phospholipid that can act on phospholipase D. Many types of lyso-type phospholipids are known, and those having different fatty acid species, molecular species having an ether or burether bond, and the like are known, and these are available as commercial products. Phospholipase D is derived from higher plants such as cabbage and peanuts, and derived from microorganisms such as Streptomyces chromofuscus.Ac tinqmadula sp. .S Available as a commercial reagent, it is derived from Actinomadula sp.No. 362. CPA is synthesized very selectively by the enzyme (Japanese Patent Application Laid-Open No. 11-37032). The reaction between the lyso-type phospholipid and phospholipase D is not particularly limited as long as the enzyme can exhibit the activity. For example, the reaction is carried out at room temperature in an acetate buffer containing calcium chloride (pH about 5 to 6). The reaction is carried out under heating (preferably about 37 ° C) for about 1 to 5 hours. The generated cPA derivative can be purified by extraction, column chromatography, thin-layer chromatography (TLC), etc. according to a conventional method.

The cyclic phosphatidic acid derivative used as an active ingredient in the present invention can improve the survival rate of nerve cells and promote the elongation of nerve cells. Therefore, according to the present invention, there is provided an agent for treating and / or preventing a neurological disorder comprising the cyclic phosphatidic acid derivative as an active ingredient. The neuropathy referred to in the present specification is preferably a cranial nerve disorder, and specific examples thereof include neurodegenerative disease, stroke, cerebral infarction, dementia, and head injury. The neurodegenerative disease mentioned here is It is a disease in which cells atrophy or degenerately shed, and examples thereof include Alzheimer's disease, Alheimer's type 1 senile dementia, amyotrophic lateral sclerosis, Parkinson's disease and the like. The drug of the present invention should be provided in the form of a pharmaceutical composition containing one or more pharmaceutically acceptable excipients for pharmaceutical preparations and a cPA derivative represented by the general formula (I) which is an active ingredient. Is preferred.

The agent of the present invention can be administered in various forms, but since the main site of action is the brain, it is preferably in a form that can cross the blood-brain barrier. Such suitable administration forms may be oral administration or parenteral administration (eg, intravenous, intramuscular, subcutaneous or intradermal injection, rectal administration, transmucosal administration, etc.). Pharmaceutical compositions suitable for oral administration include, for example, tablets, granules, capsules, powders, dissolution media, suspensions, syrups, etc., and pharmaceutical compositions suitable for parenteral administration. Examples thereof include injections, drops, suppositories, and transdermal absorbents. The dosage form of the drug of the present invention is not limited thereto. Furthermore, a sustained-release preparation can be prepared by a known technique. .

The kind of the pharmaceutical additive used for producing the drug of the present invention is not particularly limited, and can be appropriately selected by those skilled in the art. For example, excipients, disintegrants or disintegration aids, binders, lubricants, coatings, bases, solubilizers or dissolution aids, dispersants, suspensions, emulsifiers, buffers, antioxidants, preservatives Agents, tonicity agents, pH adjusters, solubilizers, stabilizers and the like can be used, and the specific components used for these purposes are well known to those skilled in the art.

Pharmaceutical additives that can be used in the preparation of formulations for oral administration include, for example, excipients such as glucose, lactose, D-mannitol, starch, and crystalline cellulose; carboxymethylcellulose, starch, and carboxymethylcellulose calcium Disintegrants or disintegration aids; binders such as hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, or gelatin; lubricating agents such as magnesium stearate or talc; hydroxypropylmethylcellulose; Coating agents such as sucrose, polyethylene glycol or titanium oxide; Bases such as petrolatum, liquid paraffin, polyethylene glycol, gelatin, kaolin, glycerin, purified water, and hard fat can be used.

Pharmaceutical additives that can be used in the preparation of preparations for injection or infusion include distilled water for injection, physiological saline, propylene glycol, and other aqueous or ready-to-use dissolving agents that can constitute injections. Solubilizing agents; tonicity agents such as glucose, sodium chloride, D-mannitol, glycerin; and pharmaceutical additives such as inorganic acids, organic acids, inorganic bases or organic bases as pH regulators. be able to.

The agent of the present invention can be administered to mammals including humans.

The dose of the drug of the present invention should be appropriately adjusted according to conditions such as the patient's age, sex, body weight, symptoms, and administration route.In general, the amount of the active ingredient per adult per day The range is from 1 gZkg to about 100 Omg / kg, and preferably from 10 gZkg to about 100 mgZkg. The above dose of the drug may be administered once a day, or may be administered several times (eg, about 2 to 4 times).

The agent of the present invention can also be used in combination with other agents effective for treating or preventing neuropathy, nutrients that supply energy to cranial nerves, and the like.

As is clear from the results of Examples 1 and 2 described below, cPA itself is a substance present in the brain of mammals, and is considered to be safe for living organisms.

The contents disclosed in the specification of Japanese Patent Application No. 2001-115925, which is the application on which the priority claim of the present application is based, are all disclosed herein by reference.

The present invention will be specifically described by the following examples, but the present invention is not limited to the examples.

Example

Example 1: Biosynthesis of cPA by phospholipase D (PLD)

Example 1 demonstrates that actinomycete PLD can be used to produce cPA from lysophosphatidylcholine (LPC), and that the enzyme that produces cPA is present in mammalian brain. And revealed.

(A) Materials and methods

(A-1) Experimental material

PLD from Streotomyces chromofuscus (S. chromofuscus) and PLD from cabbage were purchased from Sigma. PLD from Actinomadura sp. No. 362 (A. sp. No. 362) was purchased from Meito Sugar Industry. 1-oleoyl LPC and lysophosphatidylserine (LPS) were purchased from Avanti Polar lipid, INC., And lysophosphatidylethanolamine. LPJI was obtained from Doosan Serdary Res. Lad. From 1-alkyl lysophosphatidylcholine (1-alkyl LPC), 1-alkenyl phosphatidylcholine '1-logenyl pnosphatidylcholine plasmalogen; 1 One alkenyl LPC) was purchased from Sigma Company 7. Kobayashi, S., et al .: Oleoyl cPA (organic synthesis) according to the method described in Tetrahedron Lett., 34, 4047-4050 (1993). oleoyl cPA) was also used.

(A-2) 1-Synthesis of BD-LPC

Fluorescently labeled LPC was prepared as a substrate for measuring cPA production activity. That is, 1—Hexadecanoyl 2 -— (1 2— (7—Nitrobenz_2—Oxa-1,3—Diazol-1—4-yl)) 1 sn—Glycephalic 1—Phosphocholin ( 2-NBD-HPC; Avanti Polar ipids, INC.) As a starting material, lipase (from Rhizopus delemer; Seikagaku Corporation) to deacylate only the first-position fatty acid, and then add Tris-HCl buffer In ( _PH = 9), the fluorescent acyl group at position 2 was transposed to position 1 and purified using high performance liquid chromatography (HPLC) to obtain 1-NBD-LPC.

(A-3) cPA generation activity measurement

As the substrate, use a mixture of 1-NBD-LPC and LPC derived from egg yolk at 1:99. (1% NBD- LPC). The enzyme was assayed in a 100 mM acetate buffer (pH = 5.6) containing lOmM calcium chloride in the presence of {μ} 1% NBD-LPC. As an enzyme source, PLD 2.2 μg / ml derived from actinomycete 5. chromofuscus or PLDl. 4 μg / ral derived from actinomycete A. sp. No. 362 was used. The reaction was performed at 30 ° C (5. chromofuscus) or 37 ° C (A. sp. No. 362). At the end of the reaction, add 0.3 M volume of 0.1 M citric acid to the reaction solution to acidify the solution, add a mixture of chloroform: methanol (2: 1) 5.4 times the volume of the reaction solution, and centrifuge (l , 400Xg, 5 minutes) to extract lipid in the lower layer. Once more, the extraction operation of a mixed solution of chloroform and methanol (2: 1) was repeated in the same manner, and the lower layers were combined and concentrated to dryness under a nitrogen stream. The obtained lipid was redissolved in a small amount of a mixed solution of chloroform: methanol (2: 1), and spotted on silica gel (Silica Gel) 60F thin-layer chromatography plate (TLC; manufactured by E. Merck) to develop solvent; Separation of lipids with a 5% aqueous sodium sulfite solution (100: 40: 12: 5) of a form mouth methanol acetic acid Z5%, and the intensity of each fluorescent spot was measured using a fluorescence image analyzer (FLA-2000). (Fuji Film Co., Ltd.).

(A-4) Structural analysis by ESI-MS / MS

The electrospray ion source was analyzed in an anion mode using an apparatus in which a quattro II (Quattro II; manufactured by Micromass) equipped with a tandem quadrupole mass spectrometer and an HPLC were connected. Using a Hewlett Packerd model 1050 HPLC pump (manufactured by Hewlett Packerd), elution was carried out with a mixture of acetonitrile methanol (1: 1) at a flow rate of 5 / xl / min. For the sample, 3 to 51 injections of lipids dissolved in a mixture of acetonitrile / methanol (1: 1) containing 0.1% ammonium formate and a concentration of 10 to 50 ρπιο1 / μ1 were injected. Formic acid and ammonia act as donors and acceptors of the proton, respectively, when the sample is ionized. The interface between the HPLC and the MS was maintained at 80 ° C, and the nitrogen gas for evaporating the solvent was set at a pressure of 40 psi and a flow rate of 0.41 / min. In the MS minute column, the cone voltage is 30 eV for molecular ions, and in the MS / MS analysis, the cone voltage is 90 eV for fatty acids, and is 170 eV. Was monitored for phosphoric acid. In MS / MS analysis, a method is used in which an inert gas is introduced to create a locally high-pressure site, and molecular ions are subjected to collision induced dissociation (CID) to generate daughter ions. Combined. The collision gas used was argon (pressure 3.0 to 4.5 e- ⁴ Torr), and the collision energy was set at 150 eV.

(A-5) Stress fiber formation by addition of various lysophospholipids in NIH-3T3

NIH-3T3 mouse-derived fibroblasts were cultured in Dulbecco's Modified Eagle's Medium (DMEM) containing 10% fetal bovine serum (FBS; manufactured by Moregate). It was used for. NIH-3T3 was subcultured in 2.5 × 10 ⁴ cells in a Petri dish (10 cm diameter) covered with a 22 mm diameter cover glass, changed to a medium without FBS 24 hours later, and then cultured for 48 hours in a serum-starved state . After stimulating the cells by adding 10 μl of lysophospholipid and incubating at 37 ° C. for 30 minutes, Dulbecco contains 3.7% paraformaldehyde and 0.1% Triton Χ-100. Was fixed with PBS (Dalbecco's PBS) at room temperature for 10 minutes. Then, the cells were stained with 5 units of Zml of rhodamine phalloidin (Funakoshi) at 37 ° C for 1 hour. After the cover glass was washed three times with PBS, it was observed with a confocal laser single microscope TCS NT laser controlled scanning microscope (Control Laser Scanning Microscope; manufactured by Leica).

(A-6) Measurement of cPA-forming activity in rat brain

As a material, male Sprague-Dawley rats, 4 weeks old, were used. Rats were anesthetized with ether, decapitated, and whole brains were removed and stored at _80 ° C. A 10-fold volume of 0.32M sucrose solution was added to either the left or right half of the preserved rat brain (approx. 0.8 g), and a Polytron Homogenizer (Polytron), a power controller (Power control) 7, homogenize twice for 20 seconds I knew it. Hepes buffer (2 times the volume of this solution) was added to obtain a homogenate solution. The measurement of cPA-forming activity was performed with the following solution composition. That is, as a substrate, 40 nmol of l% NBD-LPC or a mixture of ¹⁴ C-LPC radiolabeled with carboxyl group carbon of 1-position fatty acid and unlabeled LPC at a ratio of 2:55 (120 nCi) The reaction was carried out at 37 ° C. in 100 μl of a homogenate solution in the presence of 450 μl of sodium oleate. The treatment after the completion of the reaction was in accordance with the conditions of the actinomycetes PLD assay.

(B) Result

(B-1) Production of LPA / cPA by actinomycetes PLD

Using 1% 1-NBD-LPC as a substrate, it was examined whether cPA generation was observed using PLDs derived from two types of actinomycetes having different phosphorylation activities. When S. chromofuscus-derived PLD was used, only 1-NBD-LPA was obtained as the main product after reaction for 20 minutes. However, when a PLD derived from A. sp. 362, which is considered to have high transphosphorylation activity, is used, a product different from LPA is mainly obtained. Product (oleoyl cPA). In order to confirm the difference between these reactions, we examined the concentration dependence of each enzyme and the time dependence of the reaction.

In the case of using PLD derived from S. chromofuscus, only LPA was generated as the LPC substrate decreased. In contrast, when PLD derived from A. sp. 362 was used, only an increase in the product corresponding to cPA was observed, and almost no LPA was produced. This indicates that different PLDs produce different products even with the same substrate. When cabbage-derived PLD with advanced enzymological analysis was used, both LPA and cPA were produced at a ratio of about 6: 4.

Here, the substrate specificity of an enzyme derived from A. sp. 362 that produces a compound equivalent to cPA was examined. The reaction was performed according to standard PLD assay conditions using 1 μl of each 1-acyl LPC, 1-alkyl LPC, 1-alkenyl LPC, LPS and LPE as a substrate. The products from each substrate were separated using TLC, the spots corresponding to LPA and cPA were scraped, and the amount of phosphorus was measured to determine the amount of production. Any substrate smell However, almost no LPA was formed. When LPC, 1-alkyl LPC, or 1-alkenyl LPC was used as a substrate, cPA was generated in a time-dependent manner, but when LPS or LPE was used as a substrate, cPA was not generated. The results showed that PLD derived from A. sp. 362 efficiently produced cPA from lysophospholipid having choline in the polar group. It was also found that alkyl and alkenyl-type LPCs could be substrates for cPA production.

(B-2) Structural analysis of PLD reaction product from A. sp. 362

To analyze the main product (compound having the same Rf value as cPA) when a 1-oleoyl LPC having oleic acid at the 1-position fatty acid is used as a substrate and reacted with PLD derived from A. sp. 362 Mass spectrometry was used. First, in order to set the conditions for mass spectrometry, 1-oleoyl cPA, which was organically synthesized as a standard, was analyzed using ESI-MS / MS in the negative ion mode. As a result, under the above conditions, a peak at m / z 417 corresponding to the molecular ion [M—H] — of l_oleoyl cPA was observed. When the PLD reaction product was analyzed under the same conditions, the peak at m / z 417 was also strongly observed. For the peak group corresponding to molecular ions, tandem mass spectrometry (MS / MS) using in-source fragmentation was performed to obtain further structural information. Daughter scan using the standard cPA molecular ion m / z 417 as a parent ion resulted in several characteristic ion peaks, which were interpreted as follows. That, m / z 281 is _{_{C 17 H 33 C00-, m /}} z 153 is _{_{[M- C 17 H 33 C0]}} _, m / z 79 corresponds to P0 ₃ _. On the other hand, the same characteristic fragment peak group was recognized as a result of the same MS / MS analysis of the ra / z 417 peak obtained by the PLD reaction. From the above, it was confirmed that the compound produced by the PLD reaction derived from A. sp. 362 was cPA.

(B-3) Formation of stress fiber in NIH-3T3 cells by PLD reaction products To further confirm that the product obtained by the PLD reaction derived from Actinomyces A sp. 362 is the same compound as cPA The biological activity was studied. In other words, the physiology of cPA One of the activities, the ability to form active stress fibers in fibroblasts, was examined. A serum-starved subconfluent state, NIH-3T3, a mouse-derived fibroblast cell line, was treated with 10 M LPA, chemically synthesized PHYLPA, or cPA, a PLD reaction product, respectively. After giving the mixture at 37 ° C for 30 minutes, the actin stress fiber was stained with rhodamine phalloidin and observed. In control cells to which no lipid was added, formation of stress fibers was not observed, whereas formation of stress fibers was observed in all three lysophospholipids used.

(B-4) cPA production in rat brain

Whether cPA producing activity was present in the rat brain was examined. As a result of a study under a plurality of different conditions, a homogenate was finally prepared with an aqueous sucrose solution (0.32 M), and oleic acid (450 // M ) When the activity was measured in the presence, spots corresponding to cPA were confirmed after incubating at 37 ° C for 60 minutes.

(C) Summary

From the above results, it was shown that cPA can be produced by a specific enzyme among the phospholipid hydrolases generally called PLD. Among the PLDs currently marketed as purified products, it was found that different products could be obtained when LPC was used as the substrate if the enzyme source was different.

In Example 1, the detection of the enzymatic activity to generate cPA from LPC in rat brain homogenate indicates that the phosphatidyl transfer reaction positively contributes to the production of bioactive lipid cPA. Is shown. It has been shown that cPA is present in the mammalian brain, and that the brain has relatively high PLD activity. LPC of the substrate, in the normal physiological activity conditions is hardly detected in the brain is believed to be generated through activation of some PLA _2.

Efficient cPA can be prepared by using PLD derived from actinomycetes A ^ p. 362 Is useful for making structural analogs of cPA. In a preparation method using such an enzyme, cPA can be prepared from 1-alkenyl LPC. 1-alkenyl LPA is detected at the time of injury to the egret corneas, has cell proliferation activity, and is thought to be involved in wound healing. In addition, it is possible to prepare the corresponding LPA PA from LPCs with different fatty acids. Example 2: Detection of cyclic phosphatidic acid from cerebrum

In Example 2, for the purpose of detecting cPA in the brain of a mammal, it was confirmed that cPA was present in the cerebrum of the maggot.

(A) Materials and methods ~

(A-1) Experimental material

The oleoyl-cPA (organic synthetic product) used as a standard product was organically synthesized according to the method described in Kobayashi, S., et al .: Tetrahedron Lett., 34, 4047-4050 (1993).ゥ Shi brain was purchased from Tokyo Shibaura Organ Co., Ltd.

(Α-2) Extraction of lipid components from brain

The presence of cPA was confirmed using the cerebrum of the Pacific Ocean as a starting material. Figure 3 summarizes the procedure. That is, 160 ml of water and 800 ml of a mixture of chlorophonolem '(methanol) (800 ml) were added to 40 g of a part of the cerebrum, homogenized, transferred to a separating funnel, stirred, and allowed to stand at room temperature. The upper and middle layers were extracted four times with 560 ml of a mixture of form and methanol (17: 3) (v / v), and the lower layer was separated. Thereafter, 80 ml of methanol and 1 M citric acid were added to the upper intermediate layer to adjust the pH to about 3, and the mixture was left as it was for 30 minutes with stirring. Further, 560 ml of a mixture of black-mouth form and methanol (17: 3) was added, and the mixture was extracted three times, and the lower layer was separated. All the resulting form layers were collected, neutralized with chloroform / methanol / 3% aqueous ammonia: 5: 1) to a pH of 7, and then dried to dryness with a nitrogen evaporator. This extract was resuspended in a mixture of form-methanol (1: 1) and used as a lipid fraction in the following purification. (A-3) Purification by thin layer chromatography (TLC)

脂質 Thin layer chromatographic extraction of lipid extract from the cerebrum using the following developing solvent system.

(Fig. 4). For separation, a silica gel 60 TLC plate (E. Merck No. 5745; manufactured by Merck & Co.) was used.

I: Form mouth methanol: 7 M ammonia water (12: 12: 1) (v / v)

Π: Black mouth form / methanolacetate Z water (25: 15: 4: 2) (v / v)

As a standard, organically synthesized oleoyl-cPA was placed on the edge of the plate, and after development, only the standard portion was colored with a Dittmer reagent, which is a phosphorus-specific color reagent. The area corresponding to the Rf value of the standard was scraped off from the plate on which the extract was placed. The lipids were extracted twice from the silica powder in the order of a mixture of form-methanol (1: 2), (1: 1) and (2: 1) using each solvent. The extracted solutions were combined, and the solvent was removed with nitrogen gas. This was resuspended in black form-methanol (1: 1), and after removing the silica gel, the purified sample was analyzed by two-dimensional TLC using the following developing solvent. For separation, a silica gel 60 TLC plate (E. Merck No. 5721; manufactured by E. Merck) was used.

III: Form mouth methanol water (60: 40: 9)

IV: Black-mouthed form Z methanolic acetate / aceton water (10: 2: 2: 4: 1) To color lipids on the plate, spray a primulin reagent that develops all lipids, and then use a fluorescent lamp with a UV lamp. Was detected and marked with a pencil. Thereafter, spots containing phospholipids were detected by spraying with Dittma reagent and quantified.

(B) Result

Purification was started from the wet cerebrum with a wet weight of 40 g. The extraction was repeated with a mixture of black-mouthed form and methanol, and a crude lipid fraction equivalent to 6.95 g was extracted. This extract lg was dissolved in 2 ml of a mixed solution of chloroform and methanol (1: 1), and 1 ml of the solution was spotted on a TLC plate having a thickness of 2 bands. TLC uses the above developing solvent systems I and II sequentially, The Rf values were in the range of 0.80 to 0.98 and 0.74 to 0.85. When this partially purified sample was analyzed by two-dimensional TLC using the developing solvent systems III and IV, spots showing the same Rf value as standard cPA were confirmed (FIG. 5 (a)).

Next, TLC using the developing solvent system III described above was performed to compare the color developed by Dittma reagent, which is a phospholipid-specific color reagent, with a standard curve created using a known amount of standard cPA. The concentration of cPA in the product

(Fig. 5 (b)). Estimating the amount of cPA described above, it means that approximately 2. lmg of cPA was purified from the cereal cerebrum with a wet weight of 40 g.

(C) Summary

In general, lipids make up 5-15% of the mammalian brain's wet weight and 65% of the dry weight, making the brain one of the most fatty organs. In Example 2, about 7 g of total lipid was extracted from 40 g of cerebrum. In addition, about 2.lmg of cPA-equivalent lipid was finally detected. Even considering the loss during purification, this is a value corresponding to less than 0.1% of the total lipid weight in the brain. Given that the proportions of phosphatidylethanolamine (PE) and phosphatidylcholine (PC), the major phospholipids in the rat brain, to total lipids are about 20% each, cPA is less than about 120% of these. Although it is a minute component equivalent to, it was proved that cPA was present in the mammalian brain. Example 3: Rings for primary cultured neurons derived from rat fetal brain ^

Action of acid (effect on survival rate and neurite)

In Example 3, we analyzed the effects of cPA on neuronal survival and neurite formation using a primary culture system derived from rat fetal brain, and found that cPA has a novel physiological activity on neurons. (Improvement of the survival rate of nerve cells and promotion of neurite outgrowth) were demonstrated.

(A) Materials and methods

(A-1) Preparation and culture of primary cultured neurons from rat fetal brain hippocampus Sprague-Dawley rats, 16 days of gestation, were used as materials. Rats were anesthetized with ether, the fetus was removed, the whole brain was removed under a stereomicroscope, and the hippocampus inside the cerebral cortex was removed. The excised hippocampus was collected in a 15 ml centrifuge tube, and the culture solution was added to 2.5% trypsin 0.5ral to make 5 ml, and the mixture was incubated at 37 ° C for 15 minutes. Then, after centrifugation at 3,000 rpm for 15 minutes, the supernatant was removed, 5 ml of the culture solution was added, and the mixture was centrifuged at 3, OOO rpm for 15 minutes. This was repeated twice more, 3 to 5 ml of the culture solution was added, pipetting was performed with a pasteur pipet, and then pipetting was performed using a syringe needle (TERUM0 NEEDLE (0.70x38); manufactured by Terumo Corporation). . Finally, the cells dissociated through a cell strainer (FALCON Cell Strainer 70 μm; manufactured by FALC0N) were subjected to primary culture using a plate. We also studied a method of coating a culture solution and a plate that can be used to culture nerve cells without causing aggregation.

The cPA derivative used in this study was synthesized according to the method described in Example 1, using oleoyl LPC as a substrate and PLD derived from Actinomyces Asp. No. 362 as an enzyme source. cPA.

(A-2) Measurement of neuronal cell viability

Primary culture was performed by the method described in (A-1), and at the same time when the cells were seeded on the plate, oleoyl cPA ^ / z ra) was added to the neurons of different densities, and after a certain time, the phase difference Using a microscope, a photograph was taken (magnification: 20 times, 5 photographs in a 1-well plate of a 24-well plate), and the viability of the cells was morphologically determined on the photograph to calculate the survival rate.

In addition, to determine the optimal concentration of cPA to increase cell viability, culture was performed at the cell density (3.0 x 10 ⁵ cells Zcm ² ) where neuron viability was highest, and 0.5, At the same time as 1.0, 5.0, or 10.1 μl of oleoyl cPA was seeded, the cells were added to the cell culture and photographed in the same manner, and the viability of the cells was determined on the photographs.

(A-3) Measurement of neurite outgrowth To investigate the effect of cPA on neurite outgrowth, primary culture was performed using the method described in (A-1), and photographs were taken at 12, 18, and 24 hours after the start of culture, and cells were taken at each time. The average length of neurites and the number of cells with neurites were analyzed using image analysis software (NIH Image 1.62).

(A— 4) Analysis of intracellular signaling system

The phosphatidylinositol 3-kinase (PI3K) inhibitors wortmannin (Wortmannin; Sigma) and LY294002 (Sigma) were dissolved in DMS0 at a concentration of 10 mM and 50 mM, respectively, and then diluted with PBS. It was added to the cell culture so that the concentration was 30ηΜ and ΙΟ ^ Μ. The inhibitor was added to the cell culture medium 2 hours before the addition of cPA to perform pretreatment. At 48 hours after the addition of cPA, a photograph of the cells was taken, and the survival rate was calculated by morphologically determining the viability of the cells on the photograph.

(B) Result

(B-1) Establishment of experimental system for primary culture conditions of neurons

As a preliminary experiment, various culture conditions were examined, and coating with nerve cells adhered to the plate, and experiments were performed with the culture solution.As a result, the culture solution was Ham's F-12, Poly-L-lysine (Poly-L-lysine) or Poly-L-ornithine (Poly-L-ornithine) was suitable for the coating. In addition, serum-free medium was used because cPA and LPA may already exist in the serum, and a growth-promoting factor was used instead of serum. As a (serum-free culture cofactor), N1 sublimation (including insulin 5 z gZml, transferrin 5 / g / ml, progesterone 20 nM, putrescine 100 / M, and sodium serous acid 30 nM; Sigma) was added.

(B-2) Effect of cPA on viability of primary cultured neurons derived from rat fetal hippocampus First, 5 μΜ of oleoyl cPA was added to neuronal cells under the culture conditions described in (B-1) above. When the cells were added to the cell culture medium, the cells survived better and neurite outgrowth was observed compared to the control without cPA. FIG. 6 shows representative cells at that time. Next, the relationship between cell density and viability was analyzed in more detail. ^{First, 3, 0X10 5, 1.0X10 7.5X10} 4 or,, 5.0 X 10 ⁴ seeded cells at a density of cells / cm ^2, the results of comparing the survival rate after 48 hours, 3.0x10 ⁵ cells ZCM ² cells Cell viability was highest when seeded at a high density (Figure 7 (a)).

Then, the cell density of 4.5 × 10 ^5, or 6. result of enhanced OX 10 ⁵ cells / cm ^2, cell viability was reduced than in the case of 3.0x10 ⁵ cells / cm ² (FIG. 7 (b)) . Therefore, it became clear that a cell density of 3.0 × 10 ⁵ cells / cm ² was suitable for cPA to most effectively increase cell viability.

Next, to determine the optimal cPA concentration for increasing cell viability, cells were seeded, and 0.5, 1.0, 5.0, or 10.3 μm of cPA was added to the medium to determine viability. . As a result, it was clarified that when the cPA concentration was 1.0 μΜ, it was most effective for cell survival (FIGS. 8 and 9).

(Β— 3) Effect of cPA on neurite outgrowth

5 μΜ of oleoyl cPA was added to the cell culture medium, and after 24 hours, 48 hours, and 72 hours, photographs were taken and analyzed using image analysis software (NIH Image 1.62), and neurites per cell were analyzed. Was analyzed for the average length. As a result, with the addition of cPA, twice the average length was observed after 24 hours compared to the control, and the effect was stronger than after 48 hours (1.5 times) and 72 hours (1.2 times). (Figure 10 (a)). Therefore, it was considered necessary to evaluate the effect of cPA on neurite outgrowth at an earlier time, and the effect of each concentration of cPA on neurite outgrowth after 12, 18, and 24 hours was examined. As a result, the neurite outgrowth at the time point of 12 hours after addition of the cPA concentration of 1.0 / zM was about 1.7 times that of the control, and showed the maximum value (FIG. 10 (b)). This value was comparable to the addition of 50 _ng / ml (optimal concentration) of NGF.

In the primary culture, neurites may be present in all neurons There is a mixture of cells with and without protrusions. Therefore, when the effect of cPA was analyzed also on the ratio of cells having neurites, the ratio of cells having neurites was higher after 24 hours in the cells to which a cPA concentration of 1.0 was added. This was a high value even when compared with the NGF-added rice cake (Figs. 11 and 12).

(B-4) Analysis of intracellular signal transduction system in survival rate increase by cPA

The phosphatidylinositol 3-kinase (PI3K) inhibitor wortmannin or LY294002 was added to the culture medium, respectively, and the survival rate was reduced to near control levels by wortmannin. In the case of LY294002, the survival rate was lower than the control value. Many of the cells were crushed and deformed. This may be the result of the cytotoxicity of LY294002n.

These results suggested that the intracellular signal transduction system via the activity of PI3K is involved in the effect of increasing the survival rate by cPA (Fig. 13).

(C) Summary

From the above results, it was clarified that at a concentration of 1. (^ M, cPA can enhance the survival rate of primary cultured rat hippocampal nervous system cells and promote neurite outgrowth. It has been clarified that cPA has a long-lasting effect on nerve cells as a long-term effect, and that cPA may promote the differentiation of nerve cells in the long term.

According to the present invention, it has been confirmed that the cPA derivative represented by the general formula (I) enhances the survival rate of neurons derived from the hippocampus of mammals and promotes neurite outgrowth. Therefore, the cPA derivative represented by the general formula (I) used in the present invention can be used for dementia, Alzheimer's disease, senile dementia of the Alzheimer's type, amyotrophic lateral sclerosis, Parkinson's disease, stroke, cerebral infarction or head. It has been clarified that it is useful as a therapeutic agent for neuropathy such as traumatic injury. According to the present invention, it is possible to increase the survival rate of nerve cells and promote neurite outgrowth. Therefore, a therapeutic and preventive agent for a neuropathy which is very effective for prevention, treatment, rehabilitation and the like of various diseases caused by death of brain nerve cells is provided.

Claims

The scope of the claims

General formula (I)

(In the formula, R is a linear or branched alkyl group having 1 to 30 carbon atoms, a linear or branched alkenyl group having 2 to 30 carbon atoms, or a linear or branched alkyl group having 2 to 30 carbon atoms. Alkynyl groups, and these groups may contain a cycloalkane ring or an aromatic ring, and 、 is a hydrogen atom or a counter cation.)

An agent for promoting the survival of nerve cells, comprising a cyclic phosphatidic acid derivative represented by the formula (1) as an active ingredient.

2. General formula (I):

(In the formula, R is a linear or branched alkyl group having 1 to 30 carbon atoms, a linear or branched alkenyl group having 2 to 30 carbon atoms, or a linear or branched alkyl group having 2 to 30 carbon atoms. Alkynyl groups, and these groups may contain a cycloalkane ring or an aromatic ring. M is a hydrogen atom or a counter cation.)

An agent for promoting elongation of nerve cells, comprising a cyclic phosphatidic acid derivative represented by the formula (1) as an active ingredient.

3. General formula (I):

An agent for treating and / or preventing a neuropathy, comprising a cyclic phosphatidic acid derivative represented by the formula (1) as an active ingredient.

4. The neurological disorder is selected from dementia, Alzheimer's disease, senile dementia of Alzheimer's type, amyotrophic lateral sclerosis, Parkinson's disease, stroke, cerebral infarction or head injury. The drug according to any one of 1 to 3.

5. The drug according to any one of claims 1 to 4, wherein the cyclic phosphatidic acid derivative represented by the general formula (I) is 1-oleoyl cyclic phosphatidic acid.